The long term objective is to understand thee mechanism of action associated with antifreeze glycoproteins (AFGP) and antifreeze polypeptides (AFP). There are at least two classes of AFGP and three distinct classes of AFP based on their different polymer length, amino acid composition, secondary structures and immunological specificities. The specific aim is to determine the three-dimensional structure of at least one protein from each class of AFGP and AFP. We will also determine the structures of some modified proteins. Component #6 from winter flounder has been crystallized. The crystals diffract to at least 1.5 A resolution and are suitable for crystallographic studies. The structure has been determined by Patterson analysis at 4.5 A. We will collect high resolution data on an area detector and extend the resolution of the map. We will locate the bound waters and examine the relationships among the hydrophilic side chains at various temperatures. We will search for the crystallization conditions for the other antifreezes and determine their structures. The role of water is crucial in all biological systems. Since all living matter contains water, the ability of the cell or organism to withstand subfreezing temperatures is a critical problem of adaptation. This freezing tolerance observed in some marine fishes has been related to the occurrence and contribution of macromolecular antifreezes. A comparative study on these proteins will enrich our knowledge of the structure, function and evolution of these proteins. Our studies of these antifreezes will be important in elucidating the architectural requirements of the protein as an effective antifreeze agent and their mechanism of action. These investigations are important to the studies of cryoinjury and have possible application in cryopreservation.